Background/Purpose: Post-translational modifications (PTMs) of self-proteins are implicated in the pathogenesis of rheumatoid arthritis (RA). The PTMs, malondialdehyde-acetaldehyde (MAA), citrulline (CIT), and the co-modification of MAA and CIT have been demonstrated to be overrepresented in RA tissues in addition to exerting robust proinflammatory and profibrotic effects on macrophages and fibroblasts. Previous studies have shown that MAA and MAA-CIT protein antigens bind to scavenger receptors on macrophages. These data showed increased binding to TLR4, SRBII and SREC. However, little is known as to whether MAA-CIT modified antigens bind additional receptors let alone which specific receptor(s) bind them. Therefore, it was the purpose of this study to determine which specific receptors bind MAA-CIT modified proteins, and whether this binding alters the morphology of the cells.

Methods: Chinese Hamster Ovary (CHO) cells transfected with a single scavenger receptor (SR): TLR2, TLR4, SRA, SRB and CD36 were incubated with 25 μg of Human serum albumin (HSA) modified with; CIT (HSA-CIT), MAA (HSA-MAA) or MAA-CIT (HSA-MAA-CIT) antigens for 90 minutes on ice. An in-cell ELISA was performed to determine binding capacity. Briefly, cells were washed and incubated with a horseradish peroxidase (HRP) conjugated anti-human albumin antibody followed by detection using TMB substrate. Reactivity was determined at 450nm and data normalized to native HSA to look for arbitrary binding units. Additionally, following exposure and incubation on ice, the cells were photographed and evaluated for morphological changes using a Nikon overhead microscope at a magnification of 20X.

Results: Binding data revealed significant increases (p< 0.0001) in MAA-CIT binding to TLR2, TLR4, SRA, and SRB compared to native HSA, HSA-CIT and HSA-MAA antigens (Figure 1). Interestingly, MAA and MAA-CIT bound equally to CD36 compared to HSA and HSA-CIT antigens suggesting MAA is the major modification resulting in binding to CD36. In other studies, the cells were imaged following incubation with MAA-CIT antigens and showed drastic morphological changes in cellular phenotype (Figure 2). Changes included increased cellular size and shape compared to control CHO cells (no SR receptor) following MAA-CIT binding to TLR4 CHO cells, with similar changes being observed with the CHO cells expressing various SRs.

Conclusion: Findings from this study demonstrate that MAA-CIT modified protein binds multiple different scavenger receptors. Results confirm previous data showing that MAA-CIT co-modified antigen binds more to TLR4, and SRB receptors than TLR2 or SRA. Interestingly, MAA and MAA-CIT bind CD36 receptors at a similar compacity. While MAA-CIT binds all the receptors at similar levels, suggesting co-modification with MAA-CIT may be a better ligand than MAA or CIT alone. Further studies are warranted to investigate these receptors on human macrophages from patients with RA and how the morphological changes observed translate to effects on cellular function.

Figure 1. Arbitrary binding unit quantification following Western Blotting of post-translationally modified HSA to scavenger receptors of the different transfected CHO cells. Control CHO-K1 cells and CHO cells expressing TLR2, TLR4, SRA, SRB, and CD36 were incubated with 25 μg of HSA, HSA-CIT, HSA-MAA, or HSA-MAA-CIT for 90 minutes at 40C. Cells were washed and binding was determined by a cellular ELISA using an HRP conjugated anti-human albumin antibody that was detected using TMB substrate measuring the absorbance at 450 nm on an ELISA reader. To quantify binding, arbitrary binding units were developed by normalizing the data to native HSA. Anova was used to compare binding across treatment groups. Comparisons to native HSA shown above the bars (**** p < 0.0001, ####p < 0.0001). HSA-MAA-CIT demonstrated statistically significant binding to TLR2, TLR4, SRA, and SRB transfected CHO cells as compared to native HSA, HSA-CIT, and HSA-MAA. In CHO cells expressing CD36, both HSA-MAA-CIT and HSA-MAA demonstrated a statistically significant increase in binding to the scavenger receptor compared to HSA and HSA-CIT N&#3f6 for each group.

Figure 2. Representative images of CHO-K1 and CHO-TLR4 cells stimulated with HSA-MAA-CIT. Following 90 minutes of incubation with 25 μg of HSA-MAA-CIT, CHO cells transfected with TLR4 scavenger receptors demonstrate marked morphological changes compared to CHO-K1 controls. CHO-K1 control cells (left) display a spindle-shaped morphology, whereas CHO-TLR4 cells (right) show a larger polygonal morphology.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/human-serum-albumin-co-modified-with-malondialdehyde-acetaldehyde-and-citrulline-bind-multiple-scavenger-receptors-and-alter-cellular-morphology/